Using RNase protection analysis, we have quantitatively evaluated the levels of MBP mRNA isoforms in vivo during and after development in different thyroidal conditions. Our results showed that the hormone is not necessary for the developmentally programmed appearance of MBP mRNA in mice but is required for optimal MBP gene expression and that thyroxine treatment reverses the effect of hypothyroidism in young rats. Nuclear run-off assay showed that T3 increases the rate of transcription of the MBP gene. We further demonstrated that rat thyroid hormone receptor alpha specifically interacts with a 32P-MBP gene fragment in the 5' flanking region (-256/+1) footprinting the region -186/-163. Moreover, the chimeric genes either -1317 MBP or -256 MBP: CAT confer thyroid hormone responsiveness when cotransfected with the receptor into NIH3T3 or NG108-15 cells. Thus our results demonstrate that the mechanism underlying thyroid hormone regulation of MBP mRNAs is at least in part at the level of transcription, mediated by a thyroid hormone response element residing within 256 bp of the 5' flanking region of the MBP gene to which the liganded receptor binds.